Each of an RAS protein and an RAF protein is a protein that forms a cascade of intracellular signaling in an RAS/RAF/MAPK pathway. In the RAS/RAF/MAPK pathway, the RAF protein is activated by an activated RAS protein, an MEK protein is activated by the activated RAF protein, and further, an MAPK protein is activated by the activated MEK protein. By this activation, cell growth and cell differentiation are controlled.
A K-ras protein that is a kind of the RAS protein is a GDP/GTP binding protein having GTPase activity. In human, the K-ras protein is encoded by a K-ras gene located on chromosome 12. It is known that the K-ras gene has a mutation in codons 12 to 13 and the like thereof (Non-Patent Documents 1-3). The mutation in the codon 12 is, in a partial sequence of SEQ ID NO: 1 in the K-ras gene, a substitution of adenine (a), cytosine (c), or thymine (t) for guanine (g) at the 220th nucleotide (n) or a substitution of adenine (a), cytosine (c), or thymine (t) for guanine (g) at the 221st nucleotide (n). The mutation in the codon 13 is, in the nucleotide sequence of SEQ ID NO: 1 in the K-ras gene, a substitution of thymine (t) for guanine (g) at the 223rd nucleotide (k) or a substitution of adenine (a) for guanine (g) at the 224th nucleotide (r). By the mutation in the codon 12, glycine (G) at the 12nd position of the K-ras protein is mutated to serine (S), arginine (R), cysteine (C), aspartic acid (D), alanine (A), valine (V), asparagine (N), phenylalanine (F), or leucine (L). By the mutation in the codon 13, glycine (G) at the 13rd position of the K-ras protein is mutated to aspartic acid (D) or cysteine (C). It has been reported that the mutation in codon 12 or 13 of the K-ras gene has connections with, for example, cancer diseases such as colonic cancer and pancreatic cancer, congenital diseases such as CFC (cardio-facio-cutaneous) and the like, and the drug resistance to an anti-EGFR antibody drug (Non-Patent Documents 1-3). Therefore, the detection of the presence or absence of these mutations in the K-ras gene, i.e., the detection of polymorphisms in the K-ras gene is very important in, for example, diagnoses of the above-mentioned diseases, selections of more effective treatment methods for the diseases, and the like.
Moreover, a BRAF protein that is a kind of the RAF protein is a protein having serine-threonine kinase activity. In human, the BRAF protein is encoded by the BRAF gene located on the chromosome 7. It has also been reported that, as well as the mutation in the K-ras gene, a mutation in the BRAF gene has connections with the above-mentioned cancer diseases, congenital diseases, and drug resistance (Non-Patent Documents 1-3). As the mutation in the BRAF gene, a substitution of adenine (a) for thymine (t) at the 229th nucleotide (w) in a partial sequence of SEQ ID NO: 2 in the BRAF gene is known. When the nucleotide is of a wild-type (t), the 600th amino acid in the BRAF protein becomes valine (V). When the nucleotide is of a mutant-type (a), the 600th amino acid in the BRAF protein becomes glutamic acid (E). It is considered that tumorigenicity is obtained by the mutation of this amino-acid residue. Therefore, the detection of the presence or absence of the mutation in the BRAF gene, i.e., the detection of a polymorphism in the BRAF gene besides the mutation in the K-ras gene makes it possible to further improve accuracy of, for example, diagnoses of the above-mentioned diseases, selections of more effective treatment methods for the diseases, and the like.
On the other hand, as a method for detecting a polymorphism in a gene, various methods have been reported. Examples thereof include a PCR (Polymerase Chain Reaction)-RFLP (Restriction Fragment Length Polymorphism) method and the like.
The PCR-RFLP method is carried out by amplifying a detection target region in a target DNA in a sample by PCR, treating the obtained amplification product with a restriction enzyme, and typing the change in restriction fragment length caused by a polymorphism according to Southern hybridization. When a target mutation is present in the gene, the recognition site of the restriction enzyme disappears. Thus, it is possible to detect the presence or absence of the mutation based on the presence or absence of cleavage, i.e., the change in restriction fragment length.
However, in the PCR-RFLP method, for example, after the PCR, it is necessary to conduct a cumbersome procedure of treating the obtained amplification product with a restriction enzyme and conducting an analysis. Furthermore, in order to treat the obtained amplification product with a restriction enzyme, the amplification product has to be temporarily taken out. Thus, there is a risk that the amplification product obtained in a first reaction may scatter and be mixed in a second reaction that is different from the first reaction. Such problems make the automation of the polymorphism detection difficult.
In light of these problems, Tm (Melting Temperature) analysis is attracting attention as a method for detecting a polymorphism in recent years. In the Tm analysis, first, using a probe complementary to a region including a detection target polymorphism, a hybrid (double-stranded nucleic acid) of a nucleic acid to be examined (hereinafter simply referred to as a “test nucleic acid”) with the probe is formed. Then, the thus-obtained hybrid is heat-treated, and dissociation (melting) of the hybrid into single-stranded nucleic acids accompanying the temperature rise is detected by measuring signals such as absorbances. By determining the Tm value based on the result of the detection, the polymorphism is determined. The Tm value becomes higher as the complementarity between the single-stranded nucleic acids of the hybrid becomes higher, and becomes lower as the complementarity between the same becomes lower. Thus, in the case where the polymorphism in a detection target site is X or Y, the Tm value of a hybrid composed of a nucleic acid containing the target polymorphism (e.g., Y) and a probe that is 100% complementary thereto is determined beforehand (the Tm value as an evaluation standard value). Subsequently, the Tm value of a hybrid composed of the test nucleic acid and the probe is measured (the Tm value as a measured value). Then, when this measured value is the same as the evaluation standard value, it can be determined that the test nucleic acid shows a perfect match with the probe, i.e., the detection target site in the test nucleic acid is the target polymorphism (Y). On the other hand, when the measured value is lower than the evaluation standard value, it can be determined that the test nucleic acid shows a mismatch with the probe, i.e., the detection target site in the test nucleic acid is the other polymorphism (X). According to such a method, a polymorphism can be detected merely by thermal-treating a PCR reaction solution containing the probe and then measuring signals, for example. Thus, it is possible to automate a detecting device.
However, in detection methods utilizing such Tm analysis, it is necessary to determine the difference in a single nucleotide from the Tm value, for example. Further, in the case where a gene has a plurality of polymorphisms, since analysis of even one sample is accompanied by a considerable amount of work, there is a problem in that the analysis of many samples is impractical. Therefore, in particular, even in the case where a wild-type polymorphism and a plurality of mutant-type polymorphisms are present together, it is required to detect the presence or absence of mutation accurately.